The overall goal of goal of this project is to determine the protective immunogenicity of a non-toxic peptide of group A streptococcal streptolysin S (SLS) and to assess its potential to enhance the efficacy of vaccines containing M and M-related proteins (Mrp). SLS is a virulence determinant of GAS that has not previously been considered as a vaccine component because the toxin is not immunogenic following GAS infection. SLS is expressed by 99% of all GAS and there is considerable evidence supporting its prominent role in the pathogenesis of GAS infections. SLS contributes to virulence of GAS by causing soft tissue damage, lysing phagocytes, mediating translocation of the organism across epithelial boundaries, facilitating iron acquisition through lysis of RBCs, acting as a quorum sensor that upregulates itself, and potentially functioning as a global regulator of other virulenc genes via untranslated mRNA. The active toxin is a 30-amino acid heterocyclic peptide that is the product of the nine-gene Sag operon. We have previously shown that a non-toxic synthetic peptide copying amino acid residues 10-30 of the SLS propeptide coupled to KLH evoked antibodies in rabbits that completely neutralized the activity of the toxin in vitro. The SLS antibodies were non-opsonic but when combined with M protein antisera, the SLS antibodies significantly enhanced phagocytosis of GAS that was mediated by M antibodies. SLS peptides have not previously been assessed for protective immunogenicity in animal models of infection. Our hypothesis is that the addition of an immunogenic peptide of SLS to M protein and Mrp-based GAS vaccines will enhance their overall efficacy by neutralizing the cytolytic toxicity of SLS, thus resulting in decreased bacterial virulence coupled with more effective antibody-mediated clearance of the organism. The aims of this proposal are: 1) To construct recombinant multivalent hybrid proteins containing N- terminal M and Mrp peptides with and without SLS peptides and to assay immune sera against both vaccine proteins for in vitro bactericidal activity against selected serotypes of GAS, and 2) To compare the protective immunogenicity of hybrid vaccine proteins containing the SLS peptide to those without the SLS peptide in mouse models of GAS infections. Of the shared virulence determinants that are considered universal GAS vaccine antigens, we hypothesize that an immunogenic peptide of SLS that evokes neutralizing antibodies has the greatest potential to impact overall vaccine efficacy by thwarting one of the prominent stealth tactics involved in streptococcal pathogenesis.